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Biomedical subjects

V Natarajan

Publications and source records attributed to V Natarajan.

At least 109 records · Page 6Linked to original sources
PubMed1989

Passive hemagglutination test for detection of antibodies to human immunodeficiency virus type 1 and comparison of the test with enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis.

A passive hemagglutination test (PHA) was developed for detecting antibodies to human immunodeficiency virus type 1 (HIV-1) utilizing sheep erythrocytes cross-linked with purified envelope glycoprotein (gp160) of HIV-1. In an analysis of 216 human serum samples, 100% correlation was observed in 86 reactive and 124 nonreactive serum samples between PHA and commercial enzyme-linked immunosorbent assays and Western blot (immunoblot) analysis. Serum samples from gp160-immunized chimpanzees also reacted equally well in PHA. The test is simple, rapid, and inexpensive, thus providing an alternate, quick method of detecting HIV antibodies. These advantages and the thermal stability of the reagents that are used make this an attractive alternative for detecting prior exposure of individuals to HIV-1.

Animals↗
PubMed1989

Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus.

A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustrating the usefulness of adenoviruses as expression vectors.

Adenoviruses, Human↗
PubMed1989

Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport.

When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gp160) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gp120 and gp41. The subunits gp120 and gp41 were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gp160 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gp120 envelope protein. Processing and transport of gp160 were blocked by the monovalent ionophore monensin, which at high concentrations (25 microM and above) was a potent inhibitor of the endoproteolytic cleavage of gp160; at lower concentrations (1 to 10 microM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 microM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gp120 that have a lower affinity for the CD4 receptor.

Biological Transport↗
PubMed1989

Mechanism of phospholipidosis in amiodarone pulmonary toxicity.

Amiodarone therapy can be complicated by life-threatening pulmonary toxicity and is invariably associated with characteristic morphologic changes in the lung consistent with a severe phospholipidosis. To determine possible mechanisms, this study utilizes a unique in vitro cell culture model of amiodarone pulmonary toxicity and demonstrates that amiodarone can directly induce an accumulation of phospholipids within bovine pulmonary artery endothelial (BPAE) cells during the first 24 hr using amiodarone concentrations equivalent to concentrations found in the blood and lungs of human subjects. For example, amiodarone at 7.5 microM during a 24-hr incubation increases [32P]orthophosphate incorporation into phospholipids from 193 +/- 10 X 10(3) dpm/10(6) cells to 266 +/- 19 X 10(3) dpm/10(6) cells (P less than .01). A generalized increase in all phospholipids occurs after amiodarone treatment of the cultured cells; however, several specific phospholipids including phosphatidylinositol, phosphatidic acid and bis(monoacylglycerol) phosphate are all significantly increased to a greater extent than other phospholipids. Furthermore, the data indicate that amiodarone is a potent inhibitor of lysosomal phospholipase A1 and A2 activities derived from the BPAE cells; whereas, amiodarone has no effect on phospholipase A1 and A2 activities from the BPAE microsomal fraction. Thus, this study demonstrates phospholipids accumulate in cultured BPAE cells exposed to amiodarone and provides direct evidence that the drug initiates a specific and nearly complete inhibition of phospholipid degradation by lysosomal phospholipase A1 and A2 suggesting a similar process may occur in vivo in the lungs of subjects with amiodarone pulmonary toxicity.

Amiodarone↗
PubMed1988

Evidence for the incorporation of [32P]orthophosphate into leaf inositol phospholipids.

Leaf discs of brinjal, tomato, sugar cane and maize rapidly incorporated [32P]orthophosphate into total phospholipids. Analyses of the labelled lipid extracts by thin-layer chromatography, autoradiography and comparison with inositol phospholipid standards demonstrated the labelling of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in addition to other phospholipids. The presence of polyphosphoinositides was further confirmed by deacylation of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and separation of the water-soluble products, glycerophosphoinositol phosphate and glycerophosphoinositol bisphosphate by formate exchange chromatography. Incorporation of [32P]orthophosphate into inositol phospholipids was time-dependent, with monoester phosphate groups attaining isotopic equilibrium within 90 min of incubation. After 2 h, incorporation of label into phosphatidylinositol, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate was about 15, 10 and 3%, respectively, of the total phospholipids. The ratio of radioactivity in phosphatidylinositol/phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate was about 5:5:1 in brinjal leaves. However, this ratio may be an overestimate of the amounts of inositol phospholipids present, as other lysophospholipids may comigrate with standards.

Animals↗
PubMed1988

Radiographic performance of two different total hip cemented arthroplasties. A survivorship analysis.

Detailed serial radiographic analysis of the femoral and acetabular components of 367 Charnley (cobalt-chrome alloy) and 383 STH (titanium alloy) primary cemented total hip prostheses was conducted. The follow-up of the Charnley prosthesis ranged from 1 to 16 years (mean follow-up, 9 years), and for the STH from 1 to 11 years (mean follow-up, 80 months). Both single period and survivorship analyses were done to evaluate the radiographic performance. The analysis revealed that the curved STH prostheses' overall radiographic performance was less satisfactory than the Charnley prostheses. However, the STH with a straight stem had significantly better radiographic performance compared with the Charnley prostheses in all parameters except in the incidence of bone-cement radiolucent lines.

Adolescent↗
PubMed1987

A rapid affinity method for isolation and characterization of sequence specific DNA binding factor.

An affinity chromatography technique using avidin monomer and biotin interaction was used for isolating a sequence specific DNA binding factor. This approach was used for purifying a transcription factor that binds to 152 to 160 base pairs upstream of the transcription start site of adenovirus IVa2 promoter. A 432 bp IVa2 promoter fragment was isolated and was incorporated with biotin at the 5' end using Bio-11-dUTP and Klenow fragment of DNA pol I, it was then bound with factor in vitro in reactions containing hela cell extract in optimized conditions. After incubation, reactions were loaded onto an avidin monomer column. Adenovirus IVa2 transcription factor was purified by 0.3M, 0.6M Nacl in binding buffer, band retardation assays demonstrated a 12,000 fold purification of factor was obtained.

Adenoviruses, Human↗
PubMed1987

Inositol phospholipid hydrolysis by rat sciatic nerve phospholipase C.

Rat sciatic nerve cytosol contains a phosphodiesterase of the phospholipase C type that catalyzes the hydrolysis of inositol phospholipids, with preferences of phosphatidylinositol 4'-phosphate (PIP) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol 4',5'-bisphosphate (PIP2), at a pH optimum of 5.5-6.0 and at maximum rates of 55, 13, and 0.7 nmol/min/mg protein, respectively. Analysis of reaction products by TLC and formate exchange chromatography shows that inositol 1,2-cyclic phosphate (83%) and diacylglycerol are the major products of PI hydrolysis. [32P]-PIP hydrolysis yields inositol bisphosphate, inositol phosphate, and inorganic phosphate, indicating the presence of phosphodiesterase, phosphomonoesterase, and/or inositol phosphate phosphatase activities in nerve cytosol. Phosphodiesterase activity is Ca2+-dependent and completely inhibited by EGTA, but phosphomonoesterase activity is independent of divalent cations or chelating agents. Phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) inhibit PI hydrolysis. They stimulate PIP and PIP2 hydrolysis up to equimolar concentrations, but are inhibitory at higher concentrations. Both diacylglycerols and free fatty acids stimulate PI hydrolysis and counteract its inhibition by PC and lysoPC. PIP2 is a poor substrate for the cytosolic phospholipase C and strongly inhibits hydrolysis of PI. However, it enhances PIP hydrolysis up to an equimolar concentration.

Animals↗
PubMed1987

Identification of a transcription factor which interacts with the distal domain of the adenovirus IVa2 promoter.

For efficient in vitro transcription from the adenovirus IVa2 promoter, two upstream control regions, one proximal to the RNA initiation site at nucleotide position (np) -39 to -48 and the other a distal domain between np -152 and -179, are necessary. By using the band competition assay of Strauss and Varshavsky (Cell 37:889, 1984), we identified a factor which binds to the IVa2 promoter. Competition studies with various deletion mutants demonstrated that the region present 152 to 160 base pairs upstream of the IVa2 transcription start site was necessary for binding of this factor. DNase I footprinting assays demonstrated directly that this factor interacted with the distal domain of the IVa2 promoter. This factor was necessary for efficient transcription from the IVa2 promoter, as evidenced by the ability of a DNA fragment containing the binding site for this factor to inhibit transcription. Its role in bidirectional activation of transcription from major late and IVa2 promoters is discussed.

Adenoviruses, Human↗
PubMed1987

Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

Adenoviruses, Human↗
PubMed1986

Adenovirus-2 E1a and E1b gene products regulate enhancer mediated transcription.

We have shown that adenovirus-2 early region 1 gene products in trans and the SV40 enhancer in cis have an additive effect in stimulating transcription from adenovirus IVa2 and major late promoters (Natarajan, V. and Salzman, N. P., Nucleic Acids Research 13, 4067, 1985). In the present study, we show that both the E1a and E1b gene products are necessary for this stimulatory effect on enhancer mediated transcription. In the absence of E1b region, the transcription is strongly suppressed by E1a. Transcription from E1a promoter is also stimulated 4-5 fold in the presence of E1b region. The data suggest that 21K protein coded by the E1b region modulates transcription from the E1a promoter and the action of E1a gene products on transcription from other promoters.

Adenovirus Early Proteins↗
PubMed1986

N-acylethanolamine phospholipid metabolism in normal and ischemic rat brain.

N-Acylethanolamine phospholipids accumulate in rat brain during post-decapitative ischemia. Small amounts of these phospholipids consisting primarily of diacyl and alkenylacyl species can be detected within 15 min of ischemia and they increase linearly for 60 min. This ischemia-induced synthesis is more pronounced in developing rat brain (approx. 5.0 nmol/h per mumol lipid P) than in adult brain (0.4 nmol). Pulse labeling experiments with subcellular preparations of 10-day-old rat brain indicate a precursor-product relationship between ethanolamine phospholipids and their N-acyl analogs. N-Acylation of endogenous substrates occurs with both microsomes and mitochondria, exhibits a pH optimum of 10 and requires 1 mM Ca2+ for maximal (0.2 mM Ca2+ for half maximal) activity. Cell-free preparations of both developing and adult rat brain contain a phosphodiesterase which hydrolyzes N-acylphosphatidylethanolamine to phosphatidic acid and N-acylethanolamine. The latter is further hydrolyzed to fatty acid and ethanolamine by an amidohydrolase. [1-3H]Ethanolamine, injected intracerebrally or intraperitoneally into 13- and 18-day-old rats, is incorporated into brain ethanolamine phospholipids. Since small amounts of radioactivity are also associated with N-acylethanolamine phospholipids 5 and 24 h after injection of the substrate, it appears that these phospholipids may occur at a very low level as a natural lipid constituent of rat brain.

Acylation↗
PubMed1986

Mapping 5' termini of JC virus late RNA.

The 5' termini of late mRNAs were mapped 17 to 19 days after primary human fetal glial cells were infected with JC virus. The major 5' start sites spanned a region of approximately 250 nucleotides, starting at nucleotide 5114, which was on the early side of the replication origin, and extending to nucleotide 242, which was on the late side of the 98-base-pair (bp) repeats. The sequence TATATAT was contained within each of the 98-bp repeats but does not specify 5' start sites in vivo. However, the sequence TACCTA, which occurred 25 to 30 bp upstream of the simian virus 40 nucleotide position 325 start site (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:625-633, 1982) and functions as a surrogate TATA box, was present 30 bp upstream of two JC virus start sites.

Base Sequence↗
PubMed1986

Mapping 5' termini of JC virus early RNAs.

Within its enhancer promoter region, the MAD-1 strain of JC virus (JCV) has two 98-base-pair tandem repeats, each containing a TATA box-like sequence. In the present study, polyadenylated early JCV mRNAs were isolated 5 or 29 days after infection of primary human fetal glial (PHFG) cells. By using S1 nuclease, the 5' termini of the early mRNAs were mapped to nucleotide position(s) (np) 122 through 125, which lies within an AT rich region (at np 113 through 127). In contrast, when JCV DNA was transcribed in vitro, we observed a single major cluster of 5' start sites at np 94 through 97, which is approximately 25 base pairs downstream from one of the TATA boxes. By day 5, the earliest time at which JCV RNA was detected, viral DNA replication had begun; it continued for at least an additional 20 days. Since more late than early RNA was present at 5 days postinfection, the early RNAs whose synthesis began at np 122 through 125 may be analogous to SV40 late early mRNA (Ghosh and Lebowitz, J. Virol. 40:224-240, 1981). However, we have not detected RNAs with 5' termini 25 to 30 bp downstream from the TATA box at earlier times. While JCV contains two identical TATA boxes, one in each of the 98-bp repeats, only the upstream TATA box functions as an early promoter element.

Cell Line↗
PubMed1985

Occurrence of N-acylethanolamine phospholipids in fish brain and spinal cord.

N-Acylethanolamine phospholipids were identified in the central nervous system of the fresh water fish, pike (Esox lucius) and carp (Cyprinus carpio), at levels ranging from 0.1 to 0.9% of total phospholipid. The N-acylethanolamine phospholipids of carp brain were isolated and characterized by chemical, biochemical and spectroscopic methods. Two major species, 1,2-diacyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 30%) and 1-O-(1'-alkenyl)-2-acyl-sn-glycero-3-phospho(N-acyl)ethanolamines (approx. 70%) were identified. The N-acyl groups of each species consisted primarily of 16:0 (approx. 60%) but also contained 16:1, 18:0 and 18:1 (approx. 10% each) and a number of trace constituents. The N-acylethanolamine phospholipids had O-acyl and O-alkenyl group compositions similar but not identical to those of the ethanolamine phospholipids of the same tissue. N-Acylethanolamine phospholipids were present in all subcellular fractions of carp brain, except mitochondria.

Animals↗
PubMed1985

Cis and trans activation of adenovirus IVa2 gene transcription.

The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter in front of a gene coding for bacterial chloramphenicol acetyl transferase (CATase) and estimating levels of CATase and IVa2 promoter specific RNA synthesized after transfection. To produce detectable amounts of CATase with the IVa2 promoter, an enhancer has to be present in cis. In the absence of enhancer sequences, the adenovirus E1A gene can not stimulate CATase synthesis. When cells were transfected with plasmids containing enhancer sequences and various IVa2 mutant promoters upstream of the CAT gene, we observed that CATase activity was not reduced significantly even after deletion of all sequences upstream of the RNA initiation site. Synthesis of IVa2 specific RNA was dependent on plasmids containing an enhancer (SV40 72 bp repeat) that was present in cis. In the absence of enhancer sequences, co-transfection to provide the adenovirus E1A gene in trans also stimulated IVa2 RNA synthesis. When HeLa cells were transfected with various deletion mutants with an enhancer in cis it was seen that sequences -38 to -64 base pairs upstream of the RNA initiation site are necessary for efficient transcription. The E1A gene in trans and an enhancer in cis have an additive effect on RNA synthesis from both IVa2 and major late promoters. The basis for the conflicting results between transcription and CATase synthesis is discussed.

Acetyltransferases↗
PubMed1985

Positive and negative control sequences within the distal domain of the adenovirus IVa2 promoter overlap with the major late promoter.

The RNA initiation sites of the adenovirus IVa2 and major late promoters (MLP) are separated by 210 base pairs and transcribed from opposite DNA strands. We had previously shown that they contained overlapping promoter sequences (V. Natarajan, M. J. Madden, and N. P. Salzman, Proc. Natl. Acad. Sci. U.S.A. 81:6290-6294, 1984). The transcription efficiencies of these two promoters were studied in vitro with templates of covalently closed circular DNAs that contained various deletion and point mutants. The distal control region of the IVa2 promoter that is located at nucleotide position (np) -152 to -242 from the RNA initiation site consists of at least two domains. The first distal domain, present between np -152 and -179, is necessary for efficient transcription of the IVa2 promoter, and it overlaps with sequences that have been shown to be necessary for efficient transcription of MLP. This region may serve as the entry site for the transcription machinery. The second distal domain consists of sequences present between np -211 and -242. These sequences are contained at the 5' end in the MLP transcript, and they inhibit transcription from the IVa2 promoter. However, these sequences are not necessary for transcription of the MLP with a covalently closed template but are needed for transcription with a linear template. The TATA box that is located at np -180 to -186 between these two domains has a critical role for efficient transcription of the MLP. A point mutation that reduces transcription from MLP by more than 80% stimulates transcription from IVa2 promoter by 10-fold. This finding is consistent with the proposal that MLP and IVa2 promoters share an entry site for transcription machinery and compete for its use.

Adenoviruses, Human↗